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31P MRS and 1H MRI of skeletal muscle have become major new tools allowing a complete non invasive investigation of muscle function both in the clinical setting and in basic research. The comparative analysis of normal and diseased muscle remains a major requirement to further define metabolic events surrounding muscle contraction and the metabolic anomalies underlying pathologies. Also, standardized rest-exercise-recovery protocols for the exploration of muscle metabolism by P-31 MRS in healthy volunteers as well as in patients with intolerance to exercise have been developed. The CRMBM protocol is based on a short-term intense exercise, which is very informative and well accepted by volunteers and patients. Invariant metabolic parameters have been defined to characterize the normal metabolic response to the protocol. Deviations from normality can be directly interpreted in terms of specific pathologies in some favorable cases. This protocol has been applied to more than 4,000 patients and healthy volunteers over a period of 15 years. On the other hand, MRI investigations provide anatomical and functional information from resting and exercising muscle. From a diagnostic point of view, dedicated pulse sequences can be used in order to detect and quantify muscle inflammation, fatty replacement, muscle hyper and hypotrophy. In most cases, MR techniques provide valuable information which has to be processed in conjunction with traditional invasive biochemical, electrophysiological and histoenzymological tests. P-31 MRS has proved particularly useful in the therapeutic follow-up of palliative therapies (coenzyme Q treatment of mitochondriopathies) and in family investigations. It is now an accepted diagnostic tool in the array of tests which are used to characterize muscle disorders in clinical routine. As a research tool, it will keep bringing new information on the physiopathology of muscle diseases in animal models and in humans and should play a role in the metabolic characterization of gene and cell therapy.

Adenosine triphosphate (ATP) has been used for more than 50 years as a major medication in our therapeutic arsenal for the termination of supraventricular tachycardia crises for which the mechanism is re-entry implicating the atrioventricular node. In the present article other applications of ATP as a diagnostic agent for several cardiac arrhythmias are described: differential diagnosis of narrow or wide QRS tachycardia, diagnosis of a latent accessory pathway (intermittent or hidden), diagnosis of intranodal duality, and non-invasive evaluation of the results of the ablation of the slow pathway, and finally non-invasive diagnosis of the tachycardia mechanism in patients with palpitations or tachycardia with an obscure mechanism.

31P magnetic resonance spectroscopy (31P MRS) is a non-invasive method to evaluate high energy compounds [adenosine triphosphate (ATP), phosphocreatin (PCr), inorganic phosphates (Pi)] and intracellular pH (pHi) of skeletal muscle during exercise and recovery. It is a clinically applicable method of investigation for severe COPD patients with respiratory failure since exercise is limited to a single group of muscle (calf). Pronounced alterations of muscular metabolism have been shown in these patients: (1) reduced aerobic capacity (as reflected by the ratio of Pi/PCr as a function of power and changes in recovery kinetics of PCr after exercise and (2) increased anaerobic metabolism (reflected by a decrease in intracellular pH). Four different studies reveal similar abnormalities. Acute oxygen administration partially improves these parameters, suggesting that other factors in addition to hypoxaemia may contribute to the metabolic impairment. The effect of increased physical activity on these abnormalities deserve further investigations.

Bioluminescence and chemiluminescence have emerged in the last decade as a major tool for biochemical and biological studies. Several very sensitive assays have been developed in our laboratory such as enzymatic assays, immunoassays and detection of nucleic acids. The use of a new instrumentation allowing to dimensional photon counting has permit the emergence of new investigations at the cellular level. In addition to the advantages of sensitivity and the real-time non-invasive nature of this detection system, the imaging potential of using low-light and photon counting video cameras has been particularly influential in establishing its ascendence over more traditional systems. This review provides a reflection in this field through several applications in life sciences.

The influence of pressure controlled reperfusion on the postischemic outcome of normal and normotensive hypertrophied rat hearts should be investigated. To induce normotensive cardiac hypertrophy, male Wistar rats received injections of isoprenaline (5 mg/kg s.c. bid for three days). Hearts were excised and perfused in the Langendorff-technique at a perfusion pressure of 75 mmHg for 30 min. After cardioplegia and 40 min of global ischemia at 25 degrees C the hearts were reperfused for 45 min. Reperfusion pressure was built up either abruptly (75 mmHg immediately) or gradually (from 40 mmHg to 75 mmHg within 30 min). Postischemic recovery was significantly affected by the mode of reperfusion in normal hearts where pressure controlled (gradual) reperfusion is superior to the abrupt reperfusion mode. In hypertrophied hearts the postischemic outcome did not differ from normal hearts if abrupt reperfusion was used, but gradual reperfusion only led to a comparably small amelioration of postischemic status.

The study of skeletal muscle by nuclear magnetic resonance (NMR) 31p in patients with chronic heart failure (CHF) or in experimental animal models has not shown metabolic abnormalities under basal conditions. However, during exercise, phosphocreatinine (PCr) depletion is increased and early intracellular acidosis has been demonstrated. These changes contribute to deterioration of exercise capacity as they are related to peak VO2 and exercise duration. Other metabolites may play an important role. The depletion of ATP at maximal exercise may be observed in some patients with severe CCF. The results of PCr recovery kinetics are contradictory. Apparently, its recovery rate is unchanged. Most biochemical and histological abnormalities are represented by a decrease in oxidative structure (type I muscular fibres, mitochondria) and in enzymatic oxidative capacity. These changes are related to the metabolic abnormalities and exercise capacity. Muscular hypotrophy alone cannot explain the metabolic changes observed in CHF. Several mechanisms could be involved, physical deconditioning probably being the most pertinent. Other factors such as neurohormonal activation and insulin resistance should be investigated. Physical training improves exercise capacity and may reverse these muscular abnormalities. A long-term benefit of physical training on morbidity and mortality should be demonstrated.

The authors developed a rapid method for determination of tolerance of streptococci and enterococci to ampicillin by measuring the bacterial TPA. Of thirty three strains from blood cultures, 11 were tolerant with a MBC/MIC ratio > 32. For these strains, the free TPA/total TPA ratio measuring the bacterial lysis after a 2hrs incubation with 2 MIC of ampicillin, had an average of 38% (SD = 21) versus 88.6% (SD = 17.9) for the non-tolerant strains. There is a good correlation between the values of MBC/MIC ratios and the free TPA/total TPA ratios. The authors concluded that TPA assessment after 2hrs incubation with antibiotic can predict the antibiotic tolerance of streptococci or enterococci.

Two cases of polymyositis were followed using phosphorus nuclear magnetic resonance spectroscopy. The spectra recorded during remission were normal, but those collected from the gastrocnemius muscle during the active phase of the diseases showed an increased inorganic phosphate level or a decreased phosphocreatine content. The intracellular pH was normal. These findings may be related to an impairement in mitochondrial metabolism secondary to the inflammatory process. Moreover, the fact that the abnormalities observed disappeared after treatment suggests that phosphorus NMR spectroscopy could be used as a non-invasive method in the follow-up of polymyositis, but this must be confirmed by further studies.

The internal concentrations of the cations H+, Na+, K+ and the intracellular ATP concentration were measured on B succinogenes adapted to the presence of the ionophore monensin (0.5 microM). Adapted bacteria were still able to regulate the intracellular concentrations of Na+ and K+, but their internal pH and ATP concentration were lower than in control bacteria.

Adenine mononucleotides of transulucent and white parts in the adductor muscle of Japanese oyster are identified and measured after the separation on PEI-cellulose column. The studies are performed on oysters kept in aerated sea water aquarium. Results obtained for both tissues are compared each other and with the literature data.

Four ultrafiltration units have been tested in order to concentrate bacteria in water. The experiments run with Escherichia coli allows the selection of an ultrafiltration apparatus which concentrates bacteria with high recovery efficiencies. The use of this concentrator is simple and fast. Its filtration power is very stable and its little capacity allows the continuous ultrafiltration of large volumes of water.

The ability of endogenous myocardial catecholamines to participate in the development of myocardial cellular alterations during a short period of severe hypoxia (30 min) was studied in isolated, Langendorff-perfused rat heart preparation, arrested by a high potassium concentration (16 mM) and perfused in the absence of exogenous substrate (Table I). Tyramine, which accelerated catecholamine depletion, also increased myocardial cell damage as assessed by a higher lactate dehydrogenase (LDH) release and a more marked reduction in cellular levels of high energy phosphates and glycogen (Table II). On the other hand, under conditions of beta-blockade (atenolol), hypoxia-induced tissular damage was reduced (Table II). These changes could be related to modifications in the cellular content of cyclic AMP (cAMP) since cAMP was consistently higher during the first 30 min of hypoxic perfusion than in control normoxic hearts (Table III) whereas cyclic GMP content remained unchanged. Moreover, interventions increasing cellular content of cAMP (theophylline, dibutyryl-cAMP) also increased hypoxic damage (Table IV), whereas N-methyl imidazole which reduced cellular content of cAMP lessened hypoxia-induced cellular alterations (Table IV). It is concluded that cellular lesions developing during the first 30 min of hypoxia in isolated arrested rat heart preparation perfused without exogenous substrate could be related to intracellular accumulation of cAMP occurring under the effect of endogenous catecholamine release.

The firefly bioluminescence ATP assay presents multiple applications in microbiological analysis. Nine ATP extraction methods were tested on thirteen Gram positive and Gram negative bacterial species. The DMSO method appears as the procedure for choice. It provides the highest yields and the best reproducibility, even at high bacterial concentrations. It is simple, rapid and can be recommended for routine analysis. Assays carried out after extraction by this procedure show that in 24 hours cultures, the ATP content ranges from 0.28 fg to 15.65 fg per cell depending on species.

Alveolar macrophages surviving in the gas phase were exposed to concentrations of NO2 (0.1, 1 and 2 ppm) for 30 minutes. The exposed cells showed morphological changes, a decrease in bactericidal activity and a reduction of the ATP content. The severity of these changes is related to NO2 concentration. This new experimental model offers new possibilities for studies on the cytotoxicity of gases.

Myocardial protection by the Hamburg oxygenated crystalloid cardioplegic solution was evaluated. A prospective metabolic study was conducted by measuring the myocardial adenosine triphosphate (ATP) and creatine phosphate (CP) contents by enzymatic techniques in 30 coronary bypass patients with a mean of 3.5 (+/- 0.9) aorto-coronary bypass grafts. Mean aortic cross clamp time was 48.4 (+/- 9.8) min. Myocardial samples were obtained from the left anterolateral ventricular wall: 1 = before CPB, 2 = before aortic cross clamp removal, 3 = 10 min following reperfusion. During ischemia, there was no statistically significant decrease in myocardial ATP contents [3.26 (+/- 0.82) vs 3.01 (+/- 0.92) mumol/g of frozen weight]; in contrast myocardial CP contents decreased significantly [2.71 (+/- 1.44) vs 1.87 (+/- 1.19) mumol/g; p = 0.01]. Following 10 min of reperfusion, the mean ATP level [2.96 (+/- 0.84) mumol/g] was 90% of the preischemic value, and myocardial CP levels (2.32 (+/- 0.92) mumol/g] increased to 85% of preischemic levels. Spontaneous myocardial defibrillation was observed in 93.3% of cases. Early postischemic myocardial function was studied in 228 cardiac operations using the same myocardial protection. 48 patients underwent multiple valve replacement (MUVR), and 180 patients had 4 or more aorto-coronary bypass grafts (CABG). Spontaneous myocardial defibrillation was observed in 90.3% of all; cases; mean CPB time after aortic cross clamp removal was 10.3 (+/- 8) min. Cardiac index by Swan-Ganz thermodilution catheter were measured just before cardiopulmonary bypass and one and twelve hours later.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects on hemoglobin oxygen transport of acute respiratory acidosis have been studied in dogs inhaling a gaseous mixture with 12% CO2 (O2 21%) for two to five hours. In a first series of experiments, it was shown that the shape of the oxyhemoglobin dissociation curve (ODC) was not modified by severe acidosis (pH congruent to 7) lasting for two and a half hours. The Hill number (N equals 2.6) did not change significantly. The aim of the second experimental series was to stuey the Bohr effect and the hemoglobin oxygen affinity (P50). The control value for the respiratory Bohr coefficient (B) was --0.54; neither after two hours (--0.52), nor after five hours of hypercapnia (--0.55) was it significantly modified. The P50 expressed at arterial pH was much increased in acidosis (congruent to 45 torr); when expressed at standard p/ 7.4, it was slightly but significantly decreased (congruent to 1 torr) at the fifth hour. At the same time there was a decrease (p smaller than 0.05) in the erythrocyte 2,3-DPG approaching 15 p. cent; on the other hand the ATP concentration did not change significantly. No significant individual correlation was found between P50(7.4), 2,3-DPG and mean hemoglobin corpuscular concentration. These results suggest that during severe respiratory acidosis neither a change in the shape of ODC, nor a change in Bohr effect do affect the hemoglobin oxygen transport. The main characteristic remains the decrease in oxygen affinity of hemoglobin, due to the erythrocyte [H+] increase induced by hypercapnia ; this phenomenon is observed as long as the 2,3-DPG decrease stays moderate.

Killing kinetics measurement is usually time-consuming and tedious. Bioluminescent adenosine-triphosphate (ATP) assay, after intracellular nucleotide release by bacterial lysis, selects very quickly normal from antibiotic-modified and dead bacteria. Two simultaneous assays are performed with more and less strong lysis reagents (nucleotide releasing bacterial NRB, nucleotide releasing somatic NRS, Lumac). Bioluminescence produced in a luciferine - luciferase system is measured with Biocounter M 2010 luminometer. Differential values of two assays reflect the intracellular ATP fraction of strongest bacteria in tested cultures. Killing curves of some beta lactamines (aminopenicillin and cephalosporins) were studied with active Escherichia coli and Streptococcus pneumoniae cultures. Bactericidal action was seen within few hours, and similar variations of intracellular ATP fraction and numbers of colony-forming units obtained by reference method were observed. This method, well-suited to large series of assays and very rapid (intracellular ATP assay within one minute), performs detailed killing kinetics in real time.

Comparative study of achromic and normally pigmented skin of three piebald patients from two families is reported here. DOPA-positive cells were not seen in the achromic areas, but the number of ATPase sites appeared to be normal; therefore the assumption of the replacement of the melanocytes by Langerhans cells cannot be supported. Through electron microscope, Langerhans cells looked strictly normal. Melanosomes appeared reduced in size and some alterations were noticed in their shape and contents. Ultrastructurally, a more prominent change consisted in increased undefined clear cells in the achromic skin. These dendritic suprabasal cells were devoid of melanosomes or Langerhans granules and might represent undifferentiated melanocytes. These features were discussed according to previous literature.

Nuclear magnetic resonance (NMR) is a non-invasive technique which, when using 31Phosphorus (as is usually the case), provides sequential determinations of the concentrations of high energy phosphate compounds and of intracellular pH in tissues subjected to electro-magnetic pulses. NMR can now be applied to the whole heart and makes it possible to record simultaneously biochemical and haemodynamic data. Referring to personal cases, the authors emphasize the value of NMR as a new device for evaluating myocardial preservation techniques during both ischaemia and reperfusion.

Measurement of intracellular ATP content by the luciferin-luciferase photometric reaction appears to be an extremely sensitive method for detecting complement dependent cytotoxicity (DCD) mediated by antibodies directed against H-2 antigens. Within the 15 min following the addition of complement, a considerable loss of ATP is observed in the antibody-coated target cells. The reaction is detectable using normal spleen lymphocytes as target cells, completely specific and much more sensitive than the classical dye exclusion test. Preliminary findings from our current study indicate that this simple and very rapid CDC method should be considered for the purpose of HLA ABC and D typing in humans.

The dynamics of cardiac adenine and uracil nucleotides, following a subcutaneous injection of isoproterenol, was studied on the rat in vivo. The effect of continuous supply of adenosine, uridine, or ribose on the level of ATP and UTP was investigated on control rats and on isoproterenol-treated animals. The precursors were administered by continuous infusion (1 ml.h-1) into the superior caval vein. 1. ATP and UTP levels were decreased within one hour after a single dose of isoproterenol (5 mg.kg-1) (Fig. 1). 2. Then, the level of ATP rose slowly toward the control value. The normal level was not reached within 48 h (Fig. 1). 3. On the contrary, the initial drop in UTP concentration was followed by a rapid restoration. The control value was reached in 3 h, and then the UTP pool was increased to 180% of the normal level, 12 h after isoproterenol application. 4. As previously shown by other authors, the restoration of ATP was accelerated by a continuous supply of adenosine (37 micromoles per hour) or ribose (170 micromoles per hour) (Fig. 2). 5. The infusion of ribose (170 micromoles per hour) or uridine (41 micromoles per hour) completely suppressed the initial decrease in UTP level caused by beta-receptor stimulation. The further enlargement of the UTP pool was greatly enhanced by ribose or uridine (Fig. 3). 6. The infusion of adenosine was also positive on UTP regeneration. On the contrary, uridine had no effect on the ATP pool (Fig. 3). 7. When supplied to non-treated animals, all precursors caused an enhancement of the UTP level. Adenosine and ribose increased the ATP pool (Fig. 2 and 3). These results contribute to the comparison of the efficiency of the various pathways of cardiac nucleotide synthesis. They show that both de novo synthesis and salvage pathways are limited by the amount of precursors. The increase in UTP synthesis caused by ribose is consistent with the theory put forward for purines (ZIMMER et GERLACH, 1974) that phosphoribosyl-pyrophosphate availability limits the efficiency of de novo synthesis of nucleotides; it demonstrates that this concept is also true for de novo synthesis of pyrimidine nucleotides.

Experiments carried out in 12 dogs with ligature of the hepatic lobar artery, show that intra-hepatic implantation of the splenic artery, or vascular pedicle, offers limited revascularisation. This produces a fall in blood flow in the implanted artery, reduction in glycogen and hepatic A.T.P., associated with a fall in the total protein content and hepatosine. At the same time, although there is a rich network of neoformed vessels, the hepatic transaminase and lactate activities undergo a pronounced rise.

Alveolar macrophages, collected by bronchopulmonary lavage and deposited on a membrane filter applied to the surface of a reservoir filled with nutrient medium, were maintained alive in direct contact with the atmosphere. Studies of morphology (using both optic and electron microscopy), of bactericidal activity and of ATP content confirmed the viability and vitality of the cell culture. This new experimental technique permits the in vitro reconstitution of the alveolar and bronchial microenvironment and offers a new method for the study of the cytotoxicity of toxic gases.

The continuous measurement of intratissular NADH concentration allows early detection of cellular respiration arrest during clinical situations, i.e. allows a non-destructive, in situ, continuous measurement of ATP formation. This detection enables the physician or surgeon to intervene during a phase of cellular respiration arrest, before structural cellular alterations occur, thereby preventing potential tissular necrosis. The method has now been validated in experimental cardiac surgery for the monitoring of myocardial preservation techniques during cardiopulmonary by-pass, and in cardiac pharmacology, for analysis of drugs' effects on myocardial energetic metabolism. Its industrial development is currently under way. Preliminary investigation strongly suggests the vast potential of this diagnostic method in both clinical and experimental fields.